Genome‐scale CRISPR–Cas9 screen identifies PAICS as a therapeutic target for EGFR wild‐type non‐small cell lung cancer

Abstract Epidermal growth factor receptor‐targeted (EGFR‐targeted) therapies show promise for non‐small cell lung cancer (NSCLC), but they are ineffective in a third of patients who lack EGFR mutations. This underlines the need for personalized treatments for patients with EGFR wild‐type NSCLC. A genome‐wide CRISPR/Cas9 screen has identified the enzyme phosphoribosylaminoimidazole carboxylase/phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS), which is vital in de novo purine biosynthesis and tumor development, as a potential drug target for EGFR wild‐type NSCLC. We have further confirmed that PAICS expression is significantly increased in NSCLC tissues and correlates with poor patient prognosis. Knockdown of PAICS resulted in a marked reduction in both in vitro and in vivo proliferation of EGFR wild‐type NSCLC cells. Additionally, PAICS silencing led to cell‐cycle arrest in these cells, with genes involved in the cell cycle pathway being differentially expressed. Consistently, an increase in cell proliferation ability and colony number was observed in cells with upregulated PAICS in EGFR wild‐type NSCLC. PAICS silencing also caused DNA damage and cell‐cycle arrest by interacting with DNA repair genes. Moreover, decreased IMPDH2 activity and activated PI3K–AKT signaling were observed in NSCLC cells with EGFR mutations, which may compromise the effectiveness of PAICS knockdown. Therefore, PAICS plays an oncogenic role in EGFR wild‐type NSCLC and represents a potential therapeutic target for this disease.


INTRODUCTION
Lung cancer is a leading cause of cancer-related mortality worldwide, and the prognosis for patients is often poor due to late-stage diagnosis. 1Over recent decades, treatment strategies for advanced NSCLC, which comprises 85% of all lung cancer cases, have undergone significant changes. 2 Targeted therapy based on small molecules has emerged as a promising approach and has substantially improved treatment outcomes for NSCLC patients carrying targetable molecular alterations, including EGFR activating mutations, anaplastic lymphoma kinase (ALK) translocations, c-ros oncogene 1 (ROS1) translocation, [3][4][5] and so on.][8][9] Osimertinib, one of the most successful EGFR tyrosine kinase inhibitors (EGFR-TKIs), has demonstrated significant improvements in median progression-free survival (PFS) and overall survival (OS) for NSCLC patients with EGFR mutations. 10,11In 2015, the United States Food and Drug Administration granted approval for the treatment of patients with EGFR T790M-mutated NSCLC.However, a considerable proportion of NSCLC patients, particularly 50% of Asian and 80% of Western patients, do not benefit from EGFR-targeted treatments unless they present with EGFR-sensitizing mutations. 12As a result, chemotherapy remains the standard of care for EGFR wild-type NSCLC patients in routine clinical practice.Hence, the development of novel personalized molecular-targeted therapies is urgently required to enhance survival and improve the quality of life for these patients.
CRISPR/Cas9-based functional genomic screening is widely utilized for systematic exploration of gene function and characterization of their role in cellular fitness on a genome-wide scale. 13This approach offers high specificity and generates stable phenotypes by creating null alleles, rather than partial gene knockdown.[16] In this study, we present genome-scale CRISPR/Cas9 fitness screens in several EGFR wild-type NSCLC cells to find genes that are essential for cell survival.As a result, PAICS, a putative bifunctional enzyme involved in histidine de novo purine biosynthesis with both 5aminoimidazole ribonucleotide carboxylase and 4-(Nsuccinylcarboxamide)−5-aminoimidazole ribonucleotide synthetase (SAICARs) activities, was identified as a new therapeutic target for EGFR wild-type NSCLC. 179][20] Nevertheless, the precise biological functions and underlying mechanisms of PAICS in EGFR wild-type NSCLC are not well established.
In the current investigation, we meticulously explored the contributory role of PAICS in the biology of EGFR wildtype NSCLC and delved into the underlying molecular mechanisms.Our analyses unequivocally demonstrated an elevated expression profile of PAICS in NSCLC histological specimens, with its augmented expression being inextricably associated with a less favorable prognostic trajectory in NSCLC patients.Notably, targeted attenuation of PAICS robustly curtailed the proliferative propensity and growth dynamics of EGFR wild-type NSCLC cellular models, both in vitro assays and in vivo murine xenograft frameworks.Of paramount significance, the silencing of PAICS orchestrated a pronounced induction of cell-cycle quiescence and incurred DNA integrity perturbations, ostensibly through its interactions with a cadre of DNA repair-centric gene entities.Comprehensive elucidation of the specific roles of PAICS in EGFR wild-type NSCLC biology would facilitate further investigations aimed at identifying potential therapeutic targets.EGFR genes, and BEAS-2B cells as control (Figure 1A).The status of some common driver gene mutations of H460, H1299, and A549 is listed in Figure 1B.Three genes were screened out according to the following procedures (Figure 1C

PAICS genetic alteration analysis data
We explored the PAICS genetic alterations in human tumor samples.The frequency of PAICS genetic alteration (∼4%) is high in Glioblastoma multiforme, Diffuse glioma and Lung squamous cell carcinoma, with "amplification" as the primary type of alteration.Melanoma and Uterine endometrioid carcinoma had the highest incidence of "mutation," with the frequency of ∼3% and ∼2%, respectively (Figure S1A).We have presented additional mutations and their locations within PAICS (Figure S1B).We did not identify a predominant type of genetic alteration, and their locations appeared to be somewhat sporadic.Among these mutations, the highest frequency was observed for the R222*/Q mutation, which occurred in five out of 189138 patients (Figure S1B).Furthermore, we identified that in cancer patients with genetic alteration of PAICS showed a poor prognosis in OS (p = 5.08e−12), PFS (p = 5.16e−3), disease-specific survival (DSS) (p = 3.003e−3), and disease-free survival (DFS) (p = 0.0131), compared with patients without PAICS alterations (Figure S1C).

PAICS is upregulated in NSCLC tissues and correlated with the prognosis of patients with NSCLC
Patient-derived tissue microarray analysis on PAICS in 83 lung adenocarcinoma samples and their corresponding adjacent normal tissues was performed.PAICS protein significantly elevated in tumor specimens revealed by IHC staining (Figures 2A and B, right).Consistent results were also observed in the analysis results of public database (Figure 2B, left).The upregulated PAICS expression was significantly associated with a poorer prognosis and a shorter OS in both public database and the microarray data (Figure 2C).At the same time, PAICS level were related to tumor metastasis, EGFR status and gender in NSCLC patients based on TCGA database (Figures 2D, E, and S2A, left), whereas uncorrelated with tumor metastasis, EGFR status and gender from microarray analysis (Figures 2D, E, and S2A, right).It is likely due to the small sample size of EGFR-positive subsamples in the microarray, which causes inaccurate statistical results.In addition, using the UAL-CAN web tool, PAICS expression were associated with TP53 mutation status and smoking habits (Figure S2B), whereas not correlated with age (Figure S2C).The above findings suggest that high level of PAICS facilitates NSCLC carcinogenesis, and the level of PAICS might be a valuable prognostic factor in NSCLC patients.

Knockdown of PAICS attenuates the malignant characteristics of tumor in vitro
To validate the screening results, the role of PAICS in NSCLC cell proliferation was investigated.PAICS in the EGFR wild-type (A549, H460, and H1299), EGFR mutant (PC9 and H1975) NSCLC cell lines and BEAS-2B cells were knockdown using short hairpin RNA-encoding lentiviruses (shNC, shPAICS#1, shPAICS#3).The status of some common driver gene mutations of PC9 and H1975 is listed in Figure S3A.Then, PAICS protein and mRNA level was successfully reduced after the shRNA transfection (Figures 3A and B).A marked reduction of the colony number was identified in PAICS knockdown A549, H460 and H1299 cells, whereas unremarkable colony number change was observed in BEAS-2B, PC9, and H1975 cells following PAICS knockdown (Figures 3C and D).Consistently, cell viability of A549, H460, and H1299 cells significantly decreased after PAICS knockdown, whereas cell viability of BEAS-2B, PC9, and H1975 showed no obvious change (Figure 3E).In addition, an increased percentage of apoptosis in PAICS-knockdown A549, H460, and H1299 cells was confirmed by flow cytometry (Figure 3F).Furthermore, we engineered an elevation of PAICS expression in BEAS-2B, H1299, A549, and H460 cellular models through meticulous transfection of PAICS-OE lentivirus.After the implementation of PAICS-OE transfection, there was a discernible augmentation in both PAICS protein and mRNA levels (Figures S3B and C).The cell viability of BEAS-2B, H1299, A549, and H460 cells exhibited a significant increment post-PAICS overexpression (Figure S3D).Consistently, a pronounced enhancement in colony number was manifested in PAICS-upregulated BEAS-2B, H1299, A549, and H460 cellular cohorts (Figures S3E and F).Moreover, based on GSEA analysis from the RNA-sequencing database, epithelial to mesenchymal transition (EMT), coagulation, and angiogenesis, the three major character-  istics related to malignant tumors, decreased in PAICS deficiency EGFR wild-type NSCLC cells (Figure 3G).To sum up, knocking down PAICS only inhibited EGFR wild-type NSCLC carcinogenesis in vitro.
Next, we identified that the level of Inosine-5′monophosphate dehydrogenase (IMPDH2), a key enzyme in the de novo purine biosynthesis pathway, is significantly lower in EGFR mutant NSCLC cells (PC9 and H1975) than EGFR wild-type NSCLC cells (H1299 and A549) (p < 0.0001;, Figure S4A).In addition, the p-AKT protein level obviously higher in EGFR mutant NSCLC cells compared with EGFR wild-type NSCLC cells after PAICS gene was knockdown (Figure S4B).Furthermore, treating EGFR mutant NSCLC cells with a pan-PI3K inhibitor BKM120 led to a significant drop of the proliferation ability of PAICS knockdown cells (Figure S4C), suggesting the activated PI3K-AKT signaling could compromise the effect of PAICS knockdown.

PAICS knockdown induces cell-cycle arrest in EGFR wild-type NSCLC cells
To explore the functions of PAICS, CancerSEA was first used for single-cell analysis.The results indicated that PAICS was primarily involved in regulating the cell cycle (Figures 4A, B, and S5A).Consistently, the KEGG pathway and GO-biological process analysis from the RNAsequencing database indicated that the downregulated genes were enriched in the regulation of the cell cycle (Figures 4C and D).Based on MSigDB (hallmark) analysis, among the nine significantly expressed hallmark gene sets, four cell cycle gene sets were enriched, G2/M checkpoint, E2F targets, mTOR signaling, and mitotic, consistent with dysregulated cell proliferation (Figure 4E).Furthermore, PAICS silencing significantly increased the proportion of cells at G-phase and reduced the proportion of cells at Sphase, suggesting that PAICS knockdown indeed blocked the cell cycle (Figure 4F).Consistently, PAICS knockdown decreased the level of the cell cycle-related genes, including CDK2, CDK6, Cyclin D1, Cyclin E, and p-Rb expression in PAICS-knockdown EGFR wild-type NSCLC cell lines (Figure 4G).Therefore, PAICS knockdown-induced cell cycle arrest might play an important role in EGFR wildtype NSCLC cells.

PAICS knockdown blocks the cell cycle by inducing DNA damage
To investigate the mechanisms of PAICS in promoting NSCLC growth, we performed RNA-seq analysis of H1299-shPAICS cells.PAICS knockdown resulted in differential expression of various genes, including 1216 upregulated genes and 1204 downregulated genes (|log2FC|≥0, Q value ≤ 0.05) (Figures 5A and B).By conducting Gene set analysis with the KEGG Pathway Term Level2 method, differentially expressed genes were enriched in DNA repair and replication pathways (Figure 5B).Consistently, differentially expressed genes were in the nucleus confirmed by GO-cellular component analysis (Figure 5C).Based on the single-cell analysis, PAICS level was significantly related to DNA damage, replication, and repair (Figures 5D and E).What is more, knockdown of PAICS led a marked increase in γH2AX foci formation in EGFR wild-type NSCLC cells (H1299, A549, and H460 cells) rather than BEAS-2B cells, which confirmed that PAICS knockdown induced DNA damage (Figures 5F and S5B).In conclusion, PAICS led to cell cycle arrest in EGFR wild-type NSCLC cells by inducing DNA damage response.

Knockdown of PAICS inhibits NSCLC carcinogenesis in vivo
The effect of PAICS on the tumor in vivo was further evaluated by establishing a subcutaneous xenograft tumor model in immunodeficient mice using PAICS-knockdown and the control H1299 cells.There was no difference in the body weight between PAICS knockdown mice and the control group (Figure S5C).As expected, tumor growth was dramatically slower in two PAICS-knockdown groups (shPAICS#1, shPAICS#3) compared with the control group (shNC) (Figures 6A and B and Tables 1 and 2).The volumes of tumor in PAICS-knockdown groups were significantly smaller, and the weights were significantly lower than those in the control group (Figures 6C and D and Tables 1  and 2).PAICS expression in subcutaneous tumor exhibited a significant decrease in PAICS knockdown group (Figure 6E).HE staining of the fixed tumor tissue showed that the nuclei were large and deeply stained, the atypia was obvious, and the pathological mitosis was common, which indicated the tumor tissue was obtained.Less aggressive and invasive growth of cells were observed in PAICS knockdown tumors compared with shNC.The front edge of the PAICS knockdown tumor was clearly visible.In contrast, the growth of the control tumor appeared to be more aggressive since a clear tumor edge was hard to be found (Figure 6F).In addition, a significant decrease was identified in the positive rate of Ki67 in the PAICSknockdown group by IHC analysis (Figures 6G and S5D).Moreover, the survival time of mice injected with H1299-shPAICS was significantly better than that of mice injected with shNC demonstrated by the survival curve (Figure 6H).Therefore, knockdown of PAICS significantly inhibited tumor growth in vivo.

Analysis of coexpressed genes correlate with PAICS in NSCLC
To investigate the PAICS coexpressed genes, we identified 4039 potential candidate genes from TCGA database.The 4039 genes were also enriched in cell cycle, DNA replication, mismatch repair, and nucleotide excision repair pathways confirmed by KEGG analysis (Figure 7A).After intersecting the above 4039 genes with 2420 differentially expressed genes related to PAICS deletion and 236 DNA damage genes from the TCGA database, five genes were acquired and ranked by p value (Figure 7B).The mRNA level of FANCI, MSH2, XRCC2, and FANCD2 indeed decreased in H1299/H460-shPAICS cells confirmed by qRT-PCR (Figure 7C).Complementarily, a notable augmentation in the mRNA expression of FANCI, MSH2, XRCC2, and FANCD2 was observed in H1299/H460-PAICS-OE cellular models (Figure S5E).At the same time, FANCI, MSH2, XRCC2, FANCD2, and GTF2H2C were used to construct a protein-protein interaction network using String (Figure 7D), which confirming FANCI, MSH2, XRCC2, and FANCD2 were correlated with PAICS again.Further bioinformatics analysis revealed FANCI, MSH2, XRCC2, and FANCD2 were positively coexpressed with PAICS (Figures 7E and F), and the upregulated expression of them was significantly associated with a poorer prognosis in NSCLC (Figure 7G).All in all, FANCI, MSH2, XRCC2, and FANCD2 were worthy of further study.Seven weeks after transplantation, eight mice were sacrificed in each group.Then, tumors were removed and weighed.

DISCUSSION
The purpose of our article is to identify therapeutic target(s) for NSCLC patients lacking EGFR-sensitizing mutations. 12This subgroup of patients currently lacks effective targeted therapy for carrying neither EGFR mutation nor other receptor tyrosine kinase mutations, somehow like triple-negative breast cancers. 21Hence, in this study, a genome-wide CRISPR/Cas9 knockout screen was performed in EGFR wild-type NSCLC and normal lung cell lines to identify therapeutic targets for this subtype of NSCLC.By means of bioinformatics analysis, three candidate targets, including PAICS, were identified as important regulators involved in the progression of EGFR wild-type NSCLC.
PAICS is a bifunctional metabolic enzyme catalyzing the conversion of AIR into SAICAR in the de novo purine biosynthesis (Figure 8). 19Biosynthesis of purine nucleotides, performed through de novo and salvage path-ways, have indispensable and diverse functions inside cells. 22De novo pathway employs simple precursors like glycine, glutamine, and aspartate to generate purine nucleotides, which is the main route in highly purineproducing tissues. 23In contrast, the salvage pathway in which nucleoside-5′-monophosphates are synthesized from purine nucleobases is important in tissues with low demand for purines. 24Rapidly dividing cancer cells rely heavily on purines derived from the de novo pathway, whereas normal cells favor the more economical and less energy-consuming salvage pathway. 25Some tumors even entirely rely on the de novo purine biosynthesis and lack the salvage pathway for adenine nucleotide synthesis. 26nzymes involved in the de novo purine biosynthesis pathway showed high activity in tumors, like dihydrofolate reductase and thymidylate synthase. 27,28Therefore, interference of enzymes catalyzing purine metabolism in the de novo pathway has been the target of rational anticancer drugs. 27he effects of PAICS are complicated and contradictory in different tumor types.0][31][32][33][34] However, a recent paper by Kobayashi et al. 29 analyzed 1659 colorectal tumor samples demonstrated a reduction of PAICS due to genetic deletion of chromosome 4q during colorectal tumor progression and metastasis.Correspondingly, loss of PAICS is also correlated with poor relapse-free survival in stage III colorectal tumor patients. 29Thus, here we set out to perform a pan-cancer analysis of PAICS.Our comprehensive analysis demonstrated that mutations in PAICS have been identified in more than 30 different types of tumors.What is more, cancer patients with genetic alteration of PAICS showed a poor prognosis in OS, PFS, DSS, and DFS.Our results indicated that PAICS expression is elevated in most tumors, with the notable exception of KIRP, where it is lower compared with respective control tissues.Taken together, these results demonstrated the critical role of PAICS in tumor progression, suggesting that PAICS is a potential biomarker for predicting the prognosis of tumor patients.However, there are no specific PAICS-targeting agents thus far.Gratifyingly, crystal structure of PAICS with CAIR/SAICAR bound in the active sites was recently revealed, disclosing important structural information for the rational design of PAICS inhibitors as potential anticancer drugs. 357][38] However, there still have no published papers to report the role and mechanism of PAICS on EGFR wild-type NSCLC, which is in a position where no targeted drugs are available.In this study, bioinformatics analysis identified that PAICS was upregulated in EGFR wild-type NSCLC cells.According to the patient-derived tissue microarray analysis in this study, PAICS expression was upregulated in the NSCLC tissue samples and particularly associated with NSCLC progression.Moreover, elevated level of PAICS was markedly related to the poor OS of NSCLC patients.All the above results suggested that PAICS plays a vital role in EGFR wild-type NSCLC progression, implying PAICS might a therapeutic target for it.
As a key regulatory enzyme involved in the de novo purine biosynthesis pathway, PAICS serves an important role in regulating cell proliferation, growth, and apoptosis.
PAICS promotes tumor cells proliferation and migration, whereas the knockdown of PAICS leads to tumor cell cycle arrest and apoptosis. 30,39In this study, shRNA-mediated PAICS knockdown showed significant suppression on the viability and proliferation, while induction on apoptosis of EGFR wild-type NSCLC cells.Moreover, PAICS knockdown resulted in the inhibition of tumor growth and improved OS in a murine xenograft model.Additionally, the augmented expression of PAICS facilitated the proliferation and growth of BEAS-2B cells and EGFR wildtype NSCLC cells.However, no noticeable changes were observed in EGFR mutation NSCLC cells following PAICS knockdown.
So, why is the suppression of PAICS expression predominantly efficacious in EGFR wild-type NSCLC?The EGFR signaling cascade is instrumental in orchestrating cellular growth and proliferation dynamics, and its inhibition markedly attenuates the proliferation potential of a diverse spectrum of neoplastic cells. 40,41We speculated that the activation of subsequent prosurvival pathways, attributed to EGFR mutations that amplify the kinase functionality of EGFR, induces a compensatory response negating the effects of PAICS suppression.For instance, activation of EGFR downstream PI3K/AKT pathway contributes to both de novo and salvage purine nucleotide synthesis. 42EGFR pathway plays a contributory role in both de novo and salvage pathways of purine nucleotide synthesis. 40,41,43Inhibition of PI3K/AKT reduced de novo synthesis by 75% in logarithmically growing C2C12 mouse mesenchymal cells. 42Analogously, the inhibition of AKT mediated by MK2296 elicited a 73% decrement in purine biosynthesis within HeLa cellular models. 44To reveal the mechanism that the EGFR wild-type NSCLC cells were more sensitive to the PAICS intervention, we performed the following experiments: first, we identified that the level of IMPDH2, a key enzyme in the de novo purine biosynthesis pathway, is significantly lower in EGFR mutant NSCLC cells than EGFR wild-type NSCLC cells.This finding suggests that EGFR wild-type NSCLC cells were more dependent on the de novo purine biosynthesis pathway than the mutant cells.Consequently, the inhibition impact of silencing PAICS was more severe on the EGFR wild-type than on the mutant NSCLC cells.Second, the PI3K/AKT signaling remained highly activated even after the PAICS gene was knockdown in EGFR mutant NSCLC cells, which may lead to the elevation of the purine salvage synthesis pathway.Furthermore, treating EGFR mutant NSCLC cells with a pan-PI3K inhibitor BKM120 leads to a significant drop of the proliferation ability of PAICS knockdown cells, suggesting the activated PI3K/AKT signaling could compromise the effect of PAICS knockdown.Therefore, we speculate that targeting PAICS is primarily effective in WT-EGFR NSCLC due to two factors: first, EGFR wild-type NSCLC cells were heavily dependent on the de novo purine synthesis pathway; second, the activation of AKT in EGFR mutant NSCLC cells compensates the blockage of de novo purine synthesis pathway by PAICS silencing.Moreover, it was further confirmed that the three major characteristics of malignant tumors (EMT, coagulation, and angiogenesis) dramatically decreased in PAICS deficiency EGFR wildtype NSCLC cells.Collectively, PAICS could be a highly promising target in EGFR wild-type NSCLC.
We further explored the detailed mechanism underlying PAICS-mediated promoting roles in tumor cell proliferation and survival.The cell cycle pathway plays a significant role in cell growth and survival. 46Aberrancy of the cell cycle is one of the most frequent alterations during tumor progression. 47Therefore, blocking cell cycle progression is regarded as an effective strategy for eliminating cancer cells. 48Some metabolic enzymes periodically translocate to the nucleus to oversee cell cycle regulators' expression. 49APDH promotes cell cycle progression by inducing advancement of the Cyclin B-CDK1 peak and delaying the degradation of telomeres. 50Pyruvate kinase M2 (PKM2) translocation accelerates cell cycle progression by inducing c-Myc expression. 51In the present study, PAICS was tightly linked to cell cycle both in single-cell analysis from CancerSEA and RNA sequencing analysis based on PAICS knockdown-caused differentially expressed genes.Four cell cycle-related gene sets (G2/M checkpoint, E2F targets, mTOR signaling and mitotic) were included among the nine significantly expressed hallmark gene sets related to PAICS.Furthermore, PAICS deficiency caused a strong arrest at the G1 phase of EGFR wild-type NSCLC cells, accompanied by the decreased expression of cell cycle factors, including CDK2, CDK6, Cyclin D1, Cyclin E, and p-Rb expression.Cyclin D-CDK4/6 complex promotes cell cycle progression in early G1 to late G1.In early G1 phase, Cyclin D-CDK4/6 phosphorylated Rb at S807/811 to release the Rb from E2F, which activates expression of genes that regulate entry into S phase. 52Cyclin E is one of E2F target genes.As Cyclin E-CDK2 phosphorylates Rb, Cyclin E reinforces its own expression through a positive feedback loop. 53Moreover, Cyclin E-CDK2 carried out a series of sequential phosphorylations to trigger DNA replication at the onset of the S phase. 54Therefore, downregulation of CDK2, CDK6, Cyclin D1, Cyclin E, and p-Rb will cause cell cycle arrest at the G1 phase, which are consistent with our results.Evidently, PAICS knockdown-induced cell cycle arrest might play a critical role in the survival of EGFR wild-type NSCLC cells.Since PKM2 is activated by SAICAR, the metabolite of PAICS, it is reasonable to assume that PAICS is involved in cell-cycle control by regulating the translocation of PKM2.
Precise DNA replication during the S phase is necessary for cell-cycle progression. 55If DNA damage is present, DNA-repair mechanisms are triggered to arrest the cell cycle until the damaged DNA is repaired. 56While severe DNA damage beyond repair will lead to cell death or senescence. 57Thus, DNA damage-induced apoptosis is a viable therapeutic target for tumors.Huang et al. have reported that PAICS participates in DNA damage repairing by targeting HDAC1-DAD51 in gastric cancer. 19In this study, PAICS deficiency induced DNA damage in EGFR wild-type NSCLC cells, which is consistent with the role of PAICS in gastric cancer.We further screened out five PAICS coexpression genes as FANCI, MSH2, XRCC2, FANCD2, and GTF2H2C using an integrated bioinformatics analysis.Further studies confirmed that there was a significant positive correlation in expression of FANCI, MSH2, XRCC2, FANCD2, and PAICS.Moreover, high level of FANCI, MSH2, XRCC2, and FANCD2 was also significantly associated with poorer prognosis in NSCLC.Therefore, we speculated that PAICS might regulate DNA damage by interacting with the FANCI, MSH2, XRCC2, and FANCD2.The detailed mechanism of PAICS in regulating FANCI, MSH2, XRCC2, and FANCD2 remains unclear and needs further exploration.
In conclusion, we successfully identified PAICS as a potential therapeutic target for EGFR wild-type NSCLC via a genome-wide CRISPR/Cas9 screen.As a novel oncogene, PAICS deficiency promotes EGFR wild-type NSCLC apoptosis by inducing DNA damage both in vitro and in vivo.These findings confirmed that PAICS is an essential gene and provided a novel and feasible therapeutic solution for NSCLC patients carrying wild-type EGFR, which are not suitable for current targeted therapies.
Overexpressed PAICS lentiviral vectors (PAICS-OE) and control group (NC) were constructed based on human PAICS sequences from the Ensembl Release 110 (July 2023) (gene nos.ENST00000505164) and synthesized by the GeneCopoeia, Inc. PAICS overexpressing sequences were cloned into pEZ-Lv242 vectors to produce PAICSoverexpressing vectors.293T cells were seeded into several six-well plates (5 × 10 5 cells/well) for 24 h before transfection.When 293T cell density reaches 60−70%, a mixture containing 1 mL of PAICS-OE/NC Plasmid and 3 µL of polybrene (1:1000) was transfected to each well of 293T cells.The cells were replenished with a fresh medium after 18 h posttransfection.After another 48 h, puro (0.5 ug/mL for BEAS-2B cells, 1 ug/mL for H1299 and A549 cells, 2 ug/mL for H460 cells) was added to the cells until a complete eradication of cells in the control group was observed.48 h postwithdrawal of puromycin, total RNA and protein were extracted from the meticulously selected cells and subjected to qRT-PCR and Western blot detection, respectively.

CRISPR/Cas9 screening with LentiCRISPRv2 library
Genome-wide CRISPR knockout screening was performed as described. 58,59As the GeCKO v2.0 library contains 123411 sgRNAs targeting 19,050 genes, about 1.6 × 10 8 NCI-H460, A549, and BEAS-2B cells need to be infected to achieve 300-fold coverage at a multiplicity of infection of 0.3-0.5.Cells were seeded in 12-well plates for 24 h and then infected with lentivirus by centrifuging at 450 g for 2 h at 37 • C in the presence of 8 µg/mL polybrene to increase transduction efficiency.After the spin, cells were replenished with fresh media (without polybrene) and cultured for another 24 h.Then, those cells were cultured in 15cm dishes to an appropriate density.Puromycin was then added into cells for 7 days at 1 µg/mL for A549 and BEAS-2B and 2 µg/mL for NCI-H460 cells.4 × 10 7 cells were harvested and stored at −80 • C for subsequent genomic DNA (gDNA) isolation (as Day 0).Meanwhile, other 4 × 10 7 cells were cultured every 3 days to maintain adequate sgRNA library complexity.On the 14th day, 4 × 10 7 cells were collected for gDNA isolation and next-generation sequencing (as Day 14).MAGeCKFlute was used to analyze and identify candidate genes.

Colony formation assay
A549, H460, H1299, BEAS-2B, PC9, and H1975 cells (500 cells/well) were seeded into 12-well plates and incubated for 14 days at 37 • C with 5% CO 2 in a humidified incubator.After incubation, the culture medium was discarded, and cells were washed with PBS.Finally, the cells were fixed with 4% paraformaldehyde and stained with a Giemsa Stain solution.Finally, all colonies were manually scored.

Cell cycle analysis and apoptosis assay
For analysis of the cell cycle, H1299, A549, and H460 cells were seeded in six-well plates and incubated with different treatments.Cells were washed twice in ice-cold PBS and fixed in 75% cold ethanol overnight at 4 • C.After removing the ethanol, the fixed cells were washed with pre-cold PBS twice, treated with 0.1 mg/mL RNase A at 37 • C in the dark for 30 min, and stained with 50 µg/mL of propidium iodide (PI) (Sigma-Aldrich, St Louis, USA) solution in the dark for 5 min.Finally, the stained cells were analyzed with a FACS Calibur flow cytometer.
For apoptosis assay, H1299, A549, and H460 cells in the logarithmic growth phase were seeded in 6-well plates, then incubated at 37 • C and 5% CO 2 condition.After incubation, the cells were rinsed with pre-cold PBS twice and then stained with Annexin V and PI according to the manufacturer's instructions.A flow cytometer was used to analyze the stained cells within 1 h.

RNA isolation and qRT-PCR
Total RNA was extracted from H460 and H1299 cells by Trizol reagent (Invitrogen) and used to synthesize cDNA with the PrimeScriptTM RT reagent Kit (TaKaRa, Dalian, China) according to the manufacturer's instructions.qRT-PCR was performed on an ABI 7900HT

Statistical analysis
Statistical analysis was performed using GraphPad Prism software (9.0;GraphPad Software, Inc.).Each experiment was repeated three times.Data are presented as the mean ± standard deviation.Unpaired Student's t-test was used for two-group comparisons, and one-way ANOVA followed by Tukey's post hoc test was used for multiple comparisons.p < 0.05 was considered to indicate a statistically significant difference.

A U T H O R C O N T R I B U T I O N S
Yufeng Li and Lingyun Zhu performed most of the experiments.Jiaqi Mao, Hongrui Zheng, Ziyi Hu, Suisui Yang, Tianyu Mao, Tingting Zhou, Pingping Cao, Hongshuai Wu, and Xuerong Wang searched the literature and conceived and supervised all the studies.Jing Wang, Fan Lin, and Hua Shen conceived and designed the research, participated in the writing and manuscript revision.All authors have read and approved the final manuscript.

A C K N O W L E D G M E N T S
This work was supported by the National Natural Science Foundation of China (32170738), the National Natural Science Youth Foundation of China (82303894), the Natural Science Foundation of Jiangsu Province (BK20211256), the Natural Science Foundation for Young Scholars of Jiangsu Province (BK20220311), and the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (21KJB180006).

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare that they have no competing interests.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

E T H I C S S TAT E M E N T
This research was conducted under the stringent oversight and approval of the Nanjing Medical University Animal Welfare and Ethics Committee (Ethical approval number: SYXK(Su)-2020-0022).

PAT I E N T C O N S E N T F O R P U B L I C AT I O N
Not applicable.

F I G U R E 1
Genome-wide CRISPR/Cas9 screens combined with the TCGA database identified PAICS as a promising therapeutic target for EGFR-WT NSCLC.(A) Schematic diagram of the CRISPR/Cas9 screens.(B) The mutated status of lung cancer driver oncogenes in H1299, A549, and H460 cell lines.(C) The flowchart shows the screening process.(D)Three genes were screened out.Survival data come from Kaplan-Meier: MOCS3(206141 -at); PAICS (214664 -at); TOMM22(217960 -s -at).

F I G U R E 2
PAICS upregulated and served as a prognostic factor in NSCLC.(A) IHC staining of PAICS and HE staining on the tissue microarray from 83 lung adenocarcinoma tissues.(B) PAICS expression in NSCLC tissues in TCGA and GTEx data (left) and microarray (right).(C) Kaplan-Meier analysis shows the OS of NSCLC patients based on PAICS expression.(D) Relationship between PAICS expression and NSCLC metastasis (left: TCGA; right: microarray).(E) Graphical representation of the relationship between the PAICS expression and NSCLC patient's EGFR status (left: TCGA; right: microarray).Data are represented as means ± SD. ****p < 0.0001, ***p < 0.001, and *p < 0.05.

F
I G U R E 4 PAICS silencing obviously induced cell-cycle arrest in EGFR-WT NSCLC cells.(A) PAICS was primarily involved in the regulation of the cell cycle in NSCLC indicated by single-cell analysis from CancerSEA.(B) PAICS mRNA level was positively correlated with cell cycle demonstrated by Data from Guillaumet-Adkins A. Genome Biol.2017 (PDX_LUAD) (n = 42).(C and D) KEGG pathway and GO-biological process analysis indicated that the cell cycle, DNA repair, and DNA replication pathway were down-regulated after knocking down PAICS.(E) MSigDB H (hallmark) analysis identified nine signatures (Q value ≤ 0.05) that were differentially expressed once silencing PAICS.(F) Cell cycle distribution of shNC-and shPAICS-NSCLC cells.(G) CDK2, CDK6, Cyclin D1, Cyclin E, and p-Rb protein levels in shNC-and shPAICS-NSCLC cells.

F
I G U R E 6 PAICS promotes NSCLC carcinogenesis in vivo.(A) The image shows xenotransplanted tumor mice.(B) Tumor volume was measured at the indicated days after mice were transplanted.(C) Xenograft tumors were removed.(D) Measure of the weight of tumor after they were removed.(E) Expression of PAICS in Subcutaneous tumors was exhibited by Western blot.(F and G) HE and Ki67 IHC staining of xenograft tumors were shown.(H) The survival time of xenografted tumor mice was monitored.
Subcutaneous tumor volume of mice.If the volume exceeds 1500 m 3 , the mice are considered dead."-" means the mouse are considered dead.
TA B L E 1Standard: